Imidazole-based synthetic lipidoids for in vivo mrna delivery into immune cells

ABSTRACT

Disclosed are lipid compounds comprising imidazole heads and lipidoid nanoparticles (LNPs) comprising the lipidoid compounds disclosed herein for efficient nucleic acid delivery to T cells.

RELATED APPLICATION

This application claims the benefit of priority to U.S. ProvisionalPatent Application Ser. No. 62/983,997, filed Mar. 2, 2020.

GOVERNMENT SUPPORT

This invention was made with government support under grants R01EB027170-01 and UG3 TR002636-01 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

BACKGROUND

Engineering T lymphocytes has tremendous potential in advancingtherapeutics of cancer, viral infections, inflammation and autoimmunity.For example, chimeric antigen receptor T cells (CARTs) has become one ofthe FDA approved lymphoma and leukemia therapies in the past fewyears 1. In current clinical strategies, intracellular delivery oftherapeutic molecules into primary T lymphocytes relies on viraldelivery system or physical methods such as electroporation. However, itrequires ex vivo enrichment of T lymphocytes, resulting in complexprocedures and high cost. Therefore, developing in vivo T cellengineering which provides time-effective and low-cost treatments isessential. mRNA is an emerging approach for cell engineering due to itsease of synthesis, rapid and transient protein expression and minimalrisk of mutagenesis. Nanomaterials, including polymer and lipidnanoparticles, have been investigated for mRNA delivery into differenttypes of cells. However, delivery of mRNA to T lymphocytes remains atechnical challenge due to limited endocytosis and protein translationof T lymphocytes. Therefore, development of better delivery system forenhanced in vivo T cell engineering is desired.

SUMMARY

In certain aspects, disclosed herein are compounds of formula I:

and pharmaceutically acceptable salts thereof, wherein

-   R⁵ is —W-L-R^(Lipid), hydrogen, halogen, amino, hydroxyl, alkoxy,    cyano, nitro, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl,    aryl, or heteroaryl; wherein one and only one of R⁵ is    —W-L-R^(Lipid).-   L is a divalent linker;-   W is NR²⁰, O, or S;-   R^(Lipid) is independently substituted or unsubstituted C₁₋₂₀ alkyl,    substituted or unsubstituted C₁₋₂₀ alkenyl, substituted or    unsubstituted C₁₋₂₀ alkynyl, substituted or unsubstituted C₁₋₂₀    heteroalkyl, substituted or unsubstituted C₁₋₂₀ heteroalkenyl, or    substituted or unsubstituted C₁₋₂₀ heteroalkynyl; and-   R²⁰ is R^(Lipid), H, C₁₋₆ alkyl, C₁₋₆ alkenyl, or C₁₋₆ alkynyl.

In certain embodiments, W is NR²⁰ or S. In certain embodiments, W is S.In certain embodiments, W is NR²⁰.

In certain embodiments, R²⁰ is R^(Lipid).

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show the optimization of lipidoid formulation, delivery timeand delivery concentration. FIG. 1A shows the luminescence expression ofprimary human CD8+T lymphocytes treated with FLuc mRNA loaded 93-O17Sformulated with different ratios of excipients. Only formulated lipidoidshowed successful delivery activity. UT: untreated. Data presented asmean±SD, n=3. FIGS. 1B-1C shows the luminescence expression of primaryhuman CD8+T lymphocytes treated with FLuc mRNA loaded 93-O17S (FIG. 1B)time dependently and (FIG. 1C) dose dependently. UT: untreated. Datapresented as mean±SD, n=2.

FIG. 2 is a schematic illustration of rough-to-detailed screening.Imidazole containing lipidoids were selected from rough screening.Imidazole and imidazole analogue containing library was constructed forthe detailed screening. Lipidoids selected from the screening were usedfor bioluminescence and gene recombination in vivo.

FIGS. 3A-3C show the rough screening of lipidoids for mRNA delivery toprimary T lymphocytes in vitro. FIG. 3A is the synthetic route oflipidoids. FIG. 3B shows the chemical structures of amine heads andcarbon tails for lipidoids synthesis. FIG. 3C shows the results of roughscreening of different lipidoid library in primary human CD8+Tlymphocytes using FLuc mRNA. “F” indicates the lipidoids formulated withcholesterol, DOPE and DSPE-PEG to the weight ratio of 16:4:1:1. “NF”indicates non-formulated lipidoids. LF2000: Lipofectamine 2000. mRNA:mRNA alone without loading to nanoparticles. Data presented as mean±SD,two separate experiments, each in triplicate.

FIGS. 4A-4B show detailed screening of imidazole and imidazole analoguehead-containing lipidoids. FIG. 4A shows the chemical structures ofamine head 93 analogue head. Amine 9310-9315 have different branch onthe spacer and different spacer length; Amine 9321-9324 have branch at2-imidazole; Amine 9331-9334 have branch at 1-imidazole and spacer at2-imidazole; Amine 9341-9352 have imidazole analogue replaced withimidazole. Structures with red indicate positive effect and blueindicate negative effect on delivery. FIG. 4B shows results of thedetailed screening of imidazole analogue heads in primary human CD8+Tlymphocytes using FLuc mRNA. UT: untreated. Data presented as mean±SD,n=3.

FIGS. 5A-5B shows detailed screening of lipidoid tails. FIG. 5A showsthe chemical structures of lipidoid with different tail. FIG. 5B showsthe results of the detailed screening of lipidoid tails in primary humanCD8+T lymphocytes using FLuc mRNA. Data presented as mean±SD, n=3.*p<0.05. **p<0.005.

FIG. 6 are graphs that shows the flow cytometry histogram of primaryhuman CD8+T lymphocytes after treated with EGFP mRNA loaded 93-O17S or9322-O17S with different concentration. UT: untreated.

FIGS. 7A-7B are bar graphs of the analysis of pKa and hemolysis oflipidoid nanoparticles. FIG. 7A. pKa analysis of lipidoid in detailedamine head 93 analogue library and tail library; FIG. 7B. Hemolysisanalysis of lipidoid in detailed amine head 93 analogue library and taillibrary.

FIGS. 8A-8C are bioluminescence images with IVIS using selectedlipidoids. FIGS. 8A-8B. Representative bioluminescence images of (FIG.8A) whole mouse and (FIG. 8B) organs after injection of FLuc mRNA loadedlipidoids intravenously. FIG. 8C. Tissues and cells were homogenized andlysed to detect luminescence expression from mice injected with FLucmRNA loaded lipidoids intravenously. Luminescence intensity wasnormalized to total protein amount. PBS: mice injected with PBS. Datapresented as mean±SD, n=3.

FIGS. 9A-9C are bioluminescence images with IVIS using ineffectivelipidoids. FIG. 9A. Representative bioluminescence images of whole mousewith IVIS after injection of FLuc mRNA loaded lipidoids intravenously.FIG. 9B. Representative bioluminescence image of organs with IVIS afterinjection of FLuc mRNA loaded 9313-O18S-S intravenously. FIG. 9C. Invivo delivery of Cre recombinase mRNA to Ai14 mice intravenously.tdTomato expression in spleen was detected by confocal microscopy 10days after the injection. T cells were labeled with CD3ε antibody.

FIGS. 10A-10C show In vivo delivery of Cre recombinase mRNA to Ai14 miceintravenously. FIGS. 10A and 10B. tdTomato expression in spleen wasdetected by confocal microscopy 10 days after the injection. T cellswere labeled with (FIG. 10A) CD3ε antibody and (FIG. 10B) CD8a antibody.FIG. 10C. Flow cytometry analysis of splenocytes 10 days after theinjection. tdTomato expression in CD4+ T cells, CD8+ T cells, B cells(CD45R), macrophages (F4/80) and dendritic cells (CD11c) werequantified. Data presented as mean±SD of three mice, each in duplicate.*p<0.05. **p<0.005. ***p<0.001.

FIG. 11 shows In vivo delivery of Cre recombinase mRNA to macrophages.tdTomato expression in spleen was detected by confocal microscopy 10days after the intravenous injection. Macrophages were labeled withF4/80 antibody.

FIG. 12 are graphs that show flow cytometry dot plot of splenocytesafter the in vivo delivery of Cre recombinase mRNA to Ai14 miceintravenously. 10 days after the delivery, tdTomato expression in CD4+ Tcells and CD8+ T cells were analyzed.

DETAILED DESCRIPTION

In certain aspects, disclosed herein are compounds of formula I:

and pharmaceutically acceptable salts thereof, wherein

-   R⁵ is —W-L-R^(Lipid), hydrogen, halogen, amino, hydroxyl, alkoxy,    cyano, nitro, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl,    aryl, or heteroaryl; wherein one and only one of R⁵ is    —W-L-R^(Lipid).-   L is a divalent linker;-   W is NR²⁰, O, or S;-   R^(Lipid) is independently substituted or unsubstituted C₁₋₂₀ alkyl,    substituted or unsubstituted C₁₋₂₀ alkenyl, substituted or    unsubstituted C₁₋₂₀ alkynyl, substituted or unsubstituted C₁₋₂₀    heteroalkyl, substituted or unsubstituted C₁₋₂₀ heteroalkenyl, or    substituted or unsubstituted C₁₋₂₀ heteroalkynyl; and-   R²⁰ is R^(Lipid), H, C₁₋₆ alkyl, C₁₋₆ alkenyl, or C₁₋₆ alkynyl.

In certain embodiments, W is NR²⁰ or S. In certain embodiments, W is S.In certain embodiments, W is NR²⁰.

In certain embodiments, R²⁰ is R^(Lipid).

In certain embodiments, R^(Lipid) is represented by formula II:

wherein

-   R¹ and R² are independently H, methyl, OH, NHR³⁰, or SH;-   R³ and R⁴ are both H; or R³ and R⁴ are taken together to form an oxo    (═O) group;-   Z is O, NR³⁰, or S;-   X and Y are independently CH₂, NR³⁰, O, S, or Se;-   m is an integer selected from 1-3;-   n is an integer selected from 1-14;-   p is 0 or 1;-   q is an integer selected from 1-10;-   t is 0 or 1; and-   R³⁰ is H, C₁₋₆ alkyl, C₁₋₆ alkenyl, or C₁₋₆ alkynyl.

In certain embodiments, R³ and R⁴ are both H. In certain embodiments, R³and R⁴ are taken together to form an oxo (═O) group.

In certain embodiments, p is 0. In certain embodiments, p is 1.

In certain embodiments, Z is O, or NR³⁰. In certain embodiments, Z is O.In certain embodiments, Z is NR³⁰.

In certain embodiments, the compound is a compound of formula III:

In certain embodiments, R¹ and R² are independently H, methyl, or OH. Incertain embodiments, R¹ and R² are both H. In certain embodiments, R¹ isH and R² is methyl. In certain embodiments, R¹ is H; and R² is OH.

In certain embodiments, X and Y are independently CH₂ or O. In certainembodiments, X and Y are both CH₂. In certain embodiments, X and Y areindependently CH₂ or O and X and Y are not the same.

In certain embodiments, X and Y are independently CH₂ or S. In certainembodiments, X and Y are both S. In certain embodiments, X and Y areindependently CH₂ or S and X and Y are not the same.

In certain embodiments, m is 1 or 2. In certain embodiments, m is 1.

In certain embodiments, n is an integer selected from 4-12. In certainembodiments, n is an integer selected from 6-10.

In certain embodiments, q is an integer selected from 2-8. In certainembodiments, q is an integer selected from 4-8.

In certain embodiments, t is 0. In certain embodiments, t is 1.

In certain embodiments, L is substituted or unsubstituted C₁₋₆ alkylene,substituted or unsubstituted C₁₋₆ alkenylene, or substituted orunsubstituted C₁₋₆ alkynylene, substituted or unsubstituted C₁₋₆heteroalkylene, substituted or unsubstituted C₁₋₆ heteroalkenylene, orsubstituted or unsubstituted C₁₋₆ heteroalkynylene.

In certain embodiments, L is substituted or unsubstituted C₁₋₆ alkylene.In certain embodiments, L is unsubstituted C₁₋₆ alkylene. In certainembodiments, L is C₁₋₆ alkylene substituted by C₁₋₆ alkyl.

In certain embodiments, L is selected from the group consisting of

In certain embodiments, R⁵ is C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl. Incertain embodiments, R⁵ is C₁₋₆ alkyl.

In certain embodiments,

is selected from the group consisting of:

In certain embodiments

is selected from the group consisting of:

In certain embodiments, each instance of R^(Lipid) is independentlyselected from the group consisting of

In certain embodiments, each instance of R^(Lipid) is independentlyselected from the group consisting of

In certain embodiments,

is selected from the group consisting of:

and each instance of R^(Lipid) is independently selected from the groupconsisting of

In another aspect, provided are lipidoid nanoparticles, comprising acompound disclosed herein.

In certain embodiments, the lipidoid nanoparticles further comprisecholesterol. In certain embodiments, the lipidoid nanoparticles furthercomprise DOPE or PEG2K-DEPC.

In certain embodiments, the lipidoid nanoparticles further comprise adivalent nickel, wherein the compound chelates with the divalent nickel.

In certain embodiments, the lipidoid nanoparticles further comprise aprotein or a nucleic acid.

In certain embodiments, the protein or the nucleic acid is GFP-Cre orCRISPR/Cas9. In certain embodiments, the protein or the nucleic acid isGFP-Cre. In certain embodiments, the protein or the nucleic acid isCRISPR/Cas9.

In certain embodiments, the divalent nickel binds to the protein or thenucleic acid via a non-covalent interaction.

In certain embodiments, the lipidoid nanoparticles further comprise asmall molecule.

In certain embodiments, the small molecule is an antifungal agent or achemotherapeutic agent.

In certain embodiments, the small molecule is selected from the groupconsisting of Bortezomib, Imatinib, Gefitinib, Erlotinib, Afatinib,Osimertinib, Dacomitinib, Daunorubicin hydrochloride, cytarabine,Fluorouracil, Irinotecan Hydrochloride, Vincristine Sulfate,Methotrexate, Paclitaxel, Vincristine Sulfate, epirubicin, docetaxel,Cyclophosphamide, Carboplatin, Lenalidomide, Ibrutinib, Abirateroneacetate, Enzalutamide, Pemetrexed, Palbociclib, Nilotinib, Everolimus,Ruxolitinib, epirubicin, pirirubicin, idarubicin, valrubicin, amrubicin,Bleomycin, phleomycin, dactinomycin, Mithramycin, streptozotecin,pentostatin, Mitosanes mitomycin C, Enediynes calicheamycin, Glycosidesrebeccamycin, Macrolide lactones epotihilones, ixabepilone, pentostatin,Salinosporamide A, Vinblastine, Vincristine, Etoposide, Teniposide,Vinorelbine, Docetaxel, Camptothecin, Hycamtin, Pederin, Theopederins,Annamides, Trabectedin, Aplidine, and Ecteinascidin 743 (ET743).

In certain embodiments, wherein the small molecule is Amphotericin B orDoxorubicin.

In certain embodiments, the lipidoid nanoparticle has a particle size ofabout 25 nm to about 1000 nm. In certain embodiments, the lipidoidnanoparticle has a particle size of about 50 nm to about 750 nm.

In yet another aspect, provided are pharmaceutical compositions,comprising a lipidoid nanoparticle disclosed herein, and apharmaceutically acceptable carrier or excipient.

Definitions

Unless otherwise defined herein, scientific and technical terms used inthis application shall have the meanings that are commonly understood bythose of ordinary skill in the art. Generally, nomenclature used inconnection with, and techniques of, chemistry, cell and tissue culture,molecular biology, cell and cancer biology, neurobiology,neurochemistry, virology, immunology, microbiology, pharmacology,genetics and protein and nucleic acid chemistry, described herein, arethose well-known and commonly used in the art.

The methods and techniques of the present disclosure are generallyperformed, unless otherwise indicated, according to conventional methodswell known in the art and as described in various general and morespecific references that are cited and discussed throughout thisspecification. See, e.g. “Principles of Neural Science”, McGraw-HillMedical, New York, N.Y. (2000); Motulsky, “Intuitive Biostatistics”,Oxford University Press, Inc. (1995); Lodish et al., “Molecular CellBiology, 4th ed.”, W. H. Freeman & Co., New York (2000); Griffiths etal., “Introduction to Genetic Analysis, 7th ed.”, W. H. Freeman & Co.,N.Y. (1999); and Gilbert et al., “Developmental Biology, 6th ed.”,Sinauer Associates, Inc., Sunderland, Mass. (2000).

Chemistry terms used herein, unless otherwise defined herein, are usedaccording to conventional usage in the art, as exemplified by “TheMcGraw-Hill Dictionary of Chemical Terms”, Parker S., Ed., McGraw-Hill,San Francisco, Calif. (1985).

As used herein, the terms “optional” or “optionally” mean that thesubsequently described event or circumstance may occur or may not occur,and that the description includes instances where the event orcircumstance occurs as well as instances in which it does not. Forexample, “optionally substituted alkyl” refers to the alkyl may besubstituted as well as where the alkyl is not substituted.

It is understood that substituents and substitution patterns on thecompounds of the present invention can be selected by one of ordinaryskilled person in the art to result chemically stable compounds whichcan be readily synthesized by techniques known in the art, as well asthose methods set forth below, from readily available startingmaterials. If a substituent is itself substituted with more than onegroup, it is understood that these multiple groups may be on the samecarbon or on different carbons, so long as a stable structure results.

As used herein, the term “optionally substituted” refers to thereplacement of one to six hydrogen radicals in a given structure withthe radical of a specified substituent including, but not limited to:hydroxyl, hydroxyalkyl, alkoxy, halogen, alkyl, nitro, silyl, acyl,acyloxy, aryl, cycloalkyl, heterocyclyl, amino, aminoalkyl, cyano,haloalkyl, haloalkoxy, —OCO—CH₂—O-alkyl, —OP(O)(O-alkyl)₂ or—CH₂—OP(O)(O-alkyl)₂. Preferably, “optionally substituted” refers to thereplacement of one to four hydrogen radicals in a given structure withthe substituents mentioned above. More preferably, one to three hydrogenradicals are replaced by the substituents as mentioned above. It isunderstood that the substituent can be further substituted.

Articles such as “a,” “an,” and “the” may mean one or more than oneunless indicated to the contrary or otherwise evident from the context.Claims or descriptions that include “or” between one or more members ofa group are considered satisfied if one, more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process unless indicated to the contrary or otherwiseevident from the context. The invention includes embodiments in whichexactly one member of the group is present in, employed in, or otherwiserelevant to a given product or process. The invention includesembodiments in which more than one, or all of the group members arepresent in, employed in, or otherwise relevant to a given product orprocess.

As used herein, the term “alkyl” refers to saturated aliphatic groups,including but not limited to C₁-C₁₀ straight-chain alkyl groups orC₁-C₁₀ branched-chain alkyl groups. Preferably, the “alkyl” group refersto C₁-C₆ straight-chain alkyl groups or C₁-C₆ branched-chain alkylgroups. Most preferably, the “alkyl” group refers to C₁-C₄straight-chain alkyl groups or C₁-C₄ branched-chain alkyl groups.Examples of “alkyl” include, but are not limited to, methyl, ethyl,1-propyl, 2-propyl, n-butyl, sec-butyl, tert-butyl, 1-pentyl, 2-pentyl,3-pentyl, neo-pentyl, 1-hexyl, 2-hexyl, 3-hexyl, 1-heptyl, 2-heptyl,3-heptyl, 4-heptyl, 1-octyl, 2-octyl, 3-octyl or 4-octyl and the like.The “alkyl” group may be optionally substituted.

The term “acyl” is art-recognized and refers to a group represented bythe general formula hydrocarbylC(O)—, preferably alkylC(O)—.

The term “acylamino” is art-recognized and refers to an amino groupsubstituted with an acyl group and may be represented, for example, bythe formula hydrocarbylC(O)NH—.

The term “acyloxy” is art-recognized and refers to a group representedby the general formula hydrocarbylC(O)O—, preferably alkylC(O)O—.

The term “alkoxy” refers to an alkyl group having an oxygen attachedthereto. Representative alkoxy groups include methoxy, ethoxy, propoxy,tert-butoxy and the like.

The term “alkoxyalkyl” refers to an alkyl group substituted with analkoxy group and may be represented by the general formulaalkyl-O-alkyl.

The term “alkyl” refers to saturated aliphatic groups, includingstraight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl(alicyclic) groups, alkyl-substituted cycloalkyl groups, andcycloalkyl-substituted alkyl groups. In preferred embodiments, astraight chain or branched chain alkyl has 30 or fewer carbon atoms inits backbone (e.g., C₁₋₃₀ for straight chains, C₃₋₃₀ for branchedchains), and more preferably 20 or fewer.

Moreover, the term “alkyl” as used throughout the specification,examples, and claims is intended to include both unsubstituted andsubstituted alkyl groups, the latter of which refers to alkyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone, including haloalkyl groups such as trifluoromethyland 2,2,2-trifluoroethyl, etc.

The term “C_(x-y)” or “C_(x)-C_(y)”, when used in conjunction with achemical moiety, such as, acyl, acyloxy, alkyl, alkenyl, alkynyl, oralkoxy is meant to include groups that contain from x to y carbons inthe chain. C₀alkyl indicates a hydrogen where the group is in a terminalposition, a bond if internal. A C₁₋₆alkyl group, for example, containsfrom one to six carbon atoms in the chain.

The term “alkylamino”, as used herein, refers to an amino groupsubstituted with at least one alkyl group.

The term “alkylthio”, as used herein, refers to a thiol groupsubstituted with an alkyl group and may be represented by the generalformula alkylS—.

The term “amide”, as used herein, refers to a group

wherein R⁹ and R¹⁰ each independently represent a hydrogen orhydrocarbyl group, or R⁹ and R¹⁰ taken together with the N atom to whichthey are attached complete a heterocycle having from 4 to 8 atoms in thering structure.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines and salts thereof, e.g., a moietythat can be represented by

wherein R⁹, R¹⁰, and R¹⁰′ each independently represent a hydrogen or ahydrocarbyl group, or R⁹ and R¹⁰ taken together with the N atom to whichthey are attached complete a heterocycle having from 4 to 8 atoms in thering structure.

The term “aminoalkyl”, as used herein, refers to an alkyl groupsubstituted with an amino group.

The term “aralkyl”, as used herein, refers to an alkyl group substitutedwith an aryl group.

The term “aryl” as used herein include substituted or unsubstitutedsingle-ring aromatic groups in which each atom of the ring is carbon.Preferably the ring is a 5- to 7-membered ring, more preferably a6-membered ring. The term “aryl” also includes polycyclic ring systemshaving two or more cyclic rings in which two or more carbons are commonto two adjoining rings wherein at least one of the rings is aromatic,e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls,cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Aryl groupsinclude benzene, naphthalene, phenanthrene, phenol, aniline, and thelike.

The term “carbamate” is art-recognized and refers to a group

wherein R⁹ and R¹⁰ independently represent hydrogen or a hydrocarbylgroup.

The term “carbocyclylalkyl”, as used herein, refers to an alkyl groupsubstituted with a carbocycle group.

The term “carbocycle” includes 5-7 membered monocyclic and 8-12 memberedbicyclic rings. Each ring of a bicyclic carbocycle may be selected fromsaturated, unsaturated and aromatic rings. Carbocycle includes bicyclicmolecules in which one, two or three or more atoms are shared betweenthe two rings. The term “fused carbocycle” refers to a bicycliccarbocycle in which each of the rings shares two adjacent atoms with theother ring. Each ring of a fused carbocycle may be selected fromsaturated, unsaturated and aromatic rings. In an exemplary embodiment,an aromatic ring, e.g., phenyl, may be fused to a saturated orunsaturated ring, e.g., cyclohexane, cyclopentane, or cyclohexene. Anycombination of saturated, unsaturated and aromatic bicyclic rings, asvalence permits, is included in the definition of carbocyclic. Exemplary“carbocycles” include cyclopentane, cyclohexane, bicyclo[2.2.1]heptane,1,5-cyclooctadiene, 1,2,3,4-tetrahydronaphthalene,bicyclo[4.2.0]oct-3-ene, naphthalene and adamantane. Exemplary fusedcarbocycles include decalin, naphthalene, 1,2,3,4-tetrahydronaphthalene,bicyclo[4.2.0]octane, 4,5,6,7-tetrahydro-1H-indene andbicyclo[4.1.0]hept-3-ene. “Carbocycles” may be substituted at any one ormore positions capable of bearing a hydrogen atom.

The term “carbocyclylalkyl”, as used herein, refers to an alkyl groupsubstituted with a carbocycle group.

The term “carbonate” is art-recognized and refers to a group —OCO₂—.

The term “carboxy”, as used herein, refers to a group represented by theformula —CO₂H.

The term “ester”, as used herein, refers to a group —C(O)OR⁹ wherein R⁹represents a hydrocarbyl group.

The term “ether”, as used herein, refers to a hydrocarbyl group linkedthrough an oxygen to another hydrocarbyl group. Accordingly, an ethersubstituent of a hydrocarbyl group may be hydrocarbyl-O—. Ethers may beeither symmetrical or unsymmetrical. Examples of ethers include, but arenot limited to, heterocycle-O-heterocycle and aryl-O-heterocycle. Ethersinclude “alkoxyalkyl” groups, which may be represented by the generalformula alkyl-O-alkyl.

The terms “halo” and “halogen” as used herein means halogen and includeschloro, fluoro, bromo, and iodo.

The terms “hetaralkyl” and “heteroaralkyl”, as used herein, refers to analkyl group substituted with a hetaryl group.

The terms “heteroaryl” and “hetaryl” include substituted orunsubstituted aromatic single ring structures, preferably 5- to7-membered rings, more preferably 5- to 6-membered rings, whose ringstructures include at least one heteroatom, preferably one to fourheteroatoms, more preferably one or two heteroatoms. The terms“heteroaryl” and “hetaryl” also include polycyclic ring systems havingtwo or more cyclic rings in which two or more carbons are common to twoadjoining rings wherein at least one of the rings is heteroaromatic,e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls,cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heteroarylgroups include, for example, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, andpyrimidine, and the like.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, andsulfur.

The term “heterocyclylalkyl”, as used herein, refers to an alkyl groupsubstituted with a heterocycle group.

The terms “heterocyclyl”, “heterocycle”, and “heterocyclic” refer tosubstituted or unsubstituted non-aromatic ring structures, preferably 3-to 10-membered rings, more preferably 3- to 7-membered rings, whose ringstructures include at least one heteroatom, preferably one to fourheteroatoms, more preferably one or two heteroatoms. The terms“heterocyclyl” and “heterocyclic” also include polycyclic ring systemshaving two or more cyclic rings in which two or more carbons are commonto two adjoining rings wherein at least one of the rings isheterocyclic, e.g., the other cyclic rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.Heterocyclyl groups include, for example, piperidine, piperazine,pyrrolidine, morpholine, lactones, lactams, and the like.

The term “hydrocarbyl”, as used herein, refers to a group that is bondedthrough a carbon atom that does not have a ═O or ═S substituent, andtypically has at least one carbon-hydrogen bond and a primarily carbonbackbone, but may optionally include heteroatoms. Thus, groups likemethyl, ethoxyethyl, 2-pyridyl, and even trifluoromethyl are consideredto be hydrocarbyl for the purposes of this application, but substituentssuch as acetyl (which has a ═O substituent on the linking carbon) andethoxy (which is linked through oxygen, not carbon) are not. Hydrocarbylgroups include, but are not limited to aryl, heteroaryl, carbocycle,heterocycle, alkyl, alkenyl, alkynyl, and combinations thereof.

The term “hydroxyalkyl”, as used herein, refers to an alkyl groupsubstituted with a hydroxy group.

The term “lower” when used in conjunction with a chemical moiety, suchas, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant toinclude groups where there are ten or fewer atoms in the substituent,preferably six or fewer. A “lower alkyl”, for example, refers to analkyl group that contains ten or fewer carbon atoms, preferably six orfewer. In certain embodiments, acyl, acyloxy, alkyl, alkenyl, alkynyl,or alkoxy substituents defined herein are respectively lower acyl, loweracyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy,whether they appear alone or in combination with other substituents,such as in the recitations hydroxyalkyl and aralkyl (in which case, forexample, the atoms within the aryl group are not counted when countingthe carbon atoms in the alkyl substituent).

The terms “polycyclyl”, “polycycle”, and “polycyclic” refer to two ormore rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls,heteroaryls, and/or heterocyclyls) in which two or more atoms are commonto two adjoining rings, e.g., the rings are “fused rings”. Each of therings of the polycycle can be substituted or unsubstituted. In certainembodiments, each ring of the polycycle contains from 3 to 10 atoms inthe ring, preferably from 5 to 7.

The term “sulfate” is art-recognized and refers to the group —OSO₃H, ora pharmaceutically acceptable salt thereof.

The term “sulfonamide” is art-recognized and refers to the grouprepresented by the general formulae

wherein R⁹ and R¹⁰ independently represents hydrogen or hydrocarbyl.

The term “sulfoxide” is art-recognized and refers to the group-S(O)—.

The term “sulfonate” is art-recognized and refers to the group SO₃H, ora pharmaceutically acceptable salt thereof.

The term “sulfone” is art-recognized and refers to the group —S(O)₂—.

The term “substituted” refers to moieties having substituents replacinga hydrogen on one or more carbons of the backbone. It will be understoodthat “substitution” or “substituted with” includes the implicit provisothat such substitution is in accordance with permitted valence of thesubstituted atom and the substituent, and that the substitution resultsin a stable compound, e.g., which does not spontaneously undergotransformation such as by rearrangement, cyclization, elimination, etc.As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and non-aromaticsubstituents of organic compounds. The permissible substituents can beone or more and the same or different for appropriate organic compounds.For purposes of this invention, the heteroatoms such as nitrogen mayhave hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valences of theheteroatoms. Substituents can include any substituents described herein,for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, analkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as athioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, aphosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine,an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, asulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, aheterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. Itwill be understood by those skilled in the art that the moietiessubstituted on the hydrocarbon chain can themselves be substituted, ifappropriate.

The term “thioalkyl”, as used herein, refers to an alkyl groupsubstituted with a thiol group.

The term “thioester”, as used herein, refers to a group —C(O)SR⁹ or—SC(O)R⁹ wherein R⁹ represents a hydrocarbyl.

The term “thioether”, as used herein, is equivalent to an ether, whereinthe oxygen is replaced with a sulfur.

The term “urea” is art-recognized and may be represented by the generalformula

wherein R⁹ and R¹⁰ independently represent hydrogen or a hydrocarbyl.

The term “modulate” as used herein includes the inhibition orsuppression of a function or activity (such as cell proliferation) aswell as the enhancement of a function or activity.

The phrase “pharmaceutically acceptable” is art-recognized. In certainembodiments, the term includes compositions, excipients, adjuvants,polymers and other materials and/or dosage forms which are, within thescope of sound medical judgment, suitable for use in contact with thetissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

“Salt” is used herein to refer to an acid addition salt or a basicaddition salt.

Many of the compounds useful in the methods and compositions of thisdisclosure have at least one stereogenic center in their structure. Thisstereogenic center may be present in a R or a S configuration, said Rand S notation is used in correspondence with the rules described inPure Appl. Chem. (1976), 45, 11-30. The disclosure contemplates allstereoisomeric forms such as enantiomeric and diastereoisomeric forms ofthe compounds, salts, prodrugs or mixtures thereof (including allpossible mixtures of stereoisomers). See, e.g., WO 01/062726.

Furthermore, certain compounds which contain alkenyl groups may exist asZ (zusammen) or E (entgegen) isomers. In each instance, the disclosureincludes both mixture and separate individual isomers.

Some of the compounds may also exist in tautomeric forms. Such forms,although not explicitly indicated in the formulae described herein, areintended to be included within the scope of the present disclosure.

“Pharmaceutically acceptable” means approved or approvable by aregulatory agency of the Federal or a state government or thecorresponding agency in countries other than the United States, or thatis listed in the U.S. Pharmacopoeia or other generally recognizedpharmacopoeia for use in animals, and more particularly, in humans.

“Pharmaceutically acceptable salt” refers to a salt of a compound of theinvention that is pharmaceutically acceptable and that possesses thedesired pharmacological activity of the parent compound. In particular,such salts are non-toxic may be inorganic or organic acid addition saltsand base addition salts. Specifically, such salts include: (1) acidaddition salts, formed with inorganic acids such as hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and thelike; or formed with organic acids such as acetic acid, propionic acid,hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid,lactic acid, malonic acid, succinic acid, malic acid, maleic acid,fumaric acid, tartaric acid, citric acid, benzoic acid,3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid,2-hydroxyethanesulfonic acid, benzenesulfonic acid,chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid,3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid,lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoicacid, salicylic acid, stearic acid, muconic acid, and the like; or (2)salts formed when an acidic proton present in the parent compound eitheris replaced by a metal ion, e.g., an alkali metal ion, an alkaline earthion, or an aluminum ion; or coordinates with an organic base such asethanolamine, diethanolamine, triethanolamine, N-methylglucamine and thelike. Salts further include, by way of example only, sodium potassium,calcium, magnesium, ammonium, tetraalkylammonium, and the like; and whenthe compound contains a basic functionality, salts of nontoxic organicor inorganic acids, such as hydrochloride, hydrobromide, tartrate,mesylate, acetate, maleate, oxalate and the like.

The term “pharmaceutically acceptable cation” refers to an acceptablecationic counterion of an acidic functional group. Such cations areexemplified by sodium, potassium, calcium, magnesium, ammonium,tetraalkylammonium cations, and the like (see, e. g., Berge, et al., J.Pharm. Sci. 66 (1):1-79 (January 77).

“Pharmaceutically acceptable vehicle” refers to a diluent, adjuvant,excipient or carrier with which a compound of the invention isadministered.

“Pharmaceutically acceptable metabolically cleavable group” refers to agroup which is cleaved in vivo to yield the parent molecule of thestructural formula indicated herein. Examples of metabolically cleavablegroups include —COR, —COOR, —CONRR and —CH₂OR radicals, where R isselected independently at each occurrence from alkyl, trialkylsilyl,carbocyclic aryl or carbocyclic aryl substituted with one or more ofalkyl, halogen, hydroxy or alkoxy. Specific examples of representativemetabolically cleavable groups include acetyl, methoxycarbonyl, benzoyl,methoxymethyl and trimethylsilyl groups.

“Prodrugs” refers to compounds, including derivatives of the compoundsof the invention, which have cleavable groups and become by solvolysisor under physiological conditions the compounds of the invention whichare pharmaceutically active in vivo. Such examples include, but are notlimited to, choline ester derivatives and the like, N-alkylmorpholineesters and the like. Other derivatives of the compounds of thisinvention have activity in both their acid and acid derivative forms,but in the acid sensitive form often offers advantages of solubility,tissue compatibility, or delayed release in the mammalian organism (see,Bundgard, H., Design of Prodrugs, pp. 7-9, 21-24, Elsevier, Amsterdam1985). Prodrugs include acid derivatives well known to practitioners ofthe art, such as, for example, esters prepared by reaction of the parentacid with a suitable alcohol, or amides prepared by reaction of theparent acid compound with a substituted or unsubstituted amine, or acidanhydrides, or mixed anhydrides. Simple aliphatic or aromatic esters,amides and anhydrides derived from acidic groups pendant on thecompounds of this invention are particular prodrugs. In some cases it isdesirable to prepare double ester type prodrugs such as(acyloxy)alkylesters or (alkoxycarbonyl)oxy)alkylesters. Particularlythe C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, aryl, C₇-C₁₂ substitutedaryl, and C₇-C₁₂ arylalkyl esters of the compounds of the invention.

“Solvate” refers to forms of the compound that are associated with asolvent or water (also referred to as “hydrate”), usually by asolvolysis reaction. This physical association includes hydrogenbonding. Conventional solvents include water, ethanol, acetic acid andthe like. The compounds of the invention may be prepared e.g., incrystalline form and may be solvated or hydrated. Suitable solvatesinclude pharmaceutically acceptable solvates, such as hydrates, andfurther include both stoichiometric solvates and non-stoichiometricsolvates. In certain instances, the solvate will be capable ofisolation, for example when one or more solvent molecules areincorporated in the crystal lattice of the crystalline solid. “Solvate”encompasses both solution-phase and isolable solvates. Representativesolvates include hydrates, ethanolates and methanolates.

A “subject” to which administration is contemplated includes, but is notlimited to, humans (i.e., a male or female of any age group, e.g., apediatric subject (e.g, infant, child, adolescent) or adult subject(e.g., young adult, middle aged adult or senior adult) and/or anon-human animal, e.g., a mammal such as primates (e.g., cynomolgusmonkeys, rhesus monkeys), cattle, pigs, horses, sheep, goats, rodents,cats, and/or dogs. In certain embodiments, the subject is a human. Incertain embodiments, the subject is a non-human animal. The terms“human,” “patient,” and “subject” are used interchangeably herein.

An “effective amount” means the amount of a compound that, whenadministered to a subject for treating or preventing a disease, issufficient to effect such treatment or prevention. The “effectiveamount” can vary depending on the compound, the disease and itsseverity, and the age, weight, etc., of the subject to be treated. A“therapeutically effective amount” refers to the effective amount fortherapeutic treatment. A “prophylactically effective amount” refers tothe effective amount for prophylactic treatment.

“Preventing” or “prevention” or “prophylactic treatment” refers to areduction in risk of acquiring or developing a disease or disorder(i.e., causing at least one of the clinical symptoms of the disease notto develop in a subject not yet exposed to a disease-causing agent, orpredisposed to the disease in advance of disease onset.

The term “prophylaxis” is related to “prevention,” and refers to ameasure or procedure the purpose of which is to prevent, rather than totreat or cure a disease. Non limiting examples of prophylactic measuresmay include the administration of vaccines; the administration of lowmolecular weight heparin to hospital patients at risk for thrombosisdue, for example, to immobilization, and the administration of ananti-malarial agent such as chloroquine, in advance of a visit to ageographical region where malaria is endemic or the risk of contractingmalaria is high.

“Treating” or “treatment” or “therapeutic treatment” of any disease ordisorder refers, in one embodiment, to ameliorating the disease ordisorder (i.e., arresting the disease or reducing the manifestation,extent or severity of at least one of the clinical symptoms thereof). Inanother embodiment “treating” or “treatment” refers to ameliorating atleast one physical parameter, which may not be discernible by thesubject. In yet another embodiment, “treating” or “treatment” refers tomodulating the disease or disorder, either physically, (e.g.,stabilization of a discernible symptom), physiologically, (e.g.,stabilization of a physical parameter), or both. In a furtherembodiment, “treating” or “treatment” relates to slowing the progressionof the disease.

As used herein, the term “isotopic variant” refers to a compound thatcontains unnatural proportions of isotopes at one or more of the atomsthat constitute such compound. For example, an “isotopic variant” of acompound can contain one or more non-radioactive isotopes, such as forexample, deuterium (²H or D), carbon-13 (¹³C), nitrogen-15 (¹⁵N), or thelike. It will be understood that, in a compound where such isotopicsubstitution is made, the following atoms, where present, may vary, sothat for example, any hydrogen may be “²H/D, any carbon may be ¹³C, orany nitrogen may be ¹⁵N, and that the presence and placement of suchatoms may be determined within the skill of the art. Likewise, theinvention may include the preparation of isotopic variants withradioisotopes, in the instance for example, where the resultingcompounds may be used for drug and/or substrate tissue distributionstudies. The radio-active isotopes tritium, i.e., ³H, and carbon-14,i.e., ¹⁴C, are particularly useful for this purpose in view of theirease of incorporation and ready means of detection. Further, compoundsmay be prepared that are substituted with positron emitting isotopes,such as ¹¹C, ¹⁸F, ¹⁵O and ¹³N, and would be useful in Positron EmissionTopography (PET) studies for examining substrate receptor occupancy. Allisotopic variants of the compounds provided herein, radioactive or not,are intended to be encompassed within the scope of the invention.

It is also to be understood that compounds that have the same molecularformula but differ in the nature or sequence of bonding of their atomsor the arrangement of their atoms in space are termed “isomers.” Isomersthat differ in the arrangement of their atoms in space are termed“stereoisomers.”

Stereoisomers that are not mirror images of one another are termed“diastereomers” and those that are non-superimposable mirror images ofeach other are termed “enantiomers.” When a compound has an asymmetriccenter, for example, it is bonded to four different groups, a pair ofenantiomers is possible. An enantiomer can be characterized by theabsolute configuration of its asymmetric center and is described by theR- and S-sequencing rules of Cahn and Prelog, or by the manner in whichthe molecule rotates the plane of polarized light and designated asdextrorotatory or levorotatory (i.e., as (+)- or (−)-isomersrespectively). A chiral compound can exist as either individualenantiomer or as a mixture thereof. A mixture containing equalproportions of the enantiomers is called a “racemic mixture”.

“Tautomers” refer to compounds that are interchangeable forms of aparticular compound structure, and that vary in the displacement ofhydrogen atoms and electrons. Thus, two structures may be in equilibriumthrough the movement of it electrons and an atom (usually H). Forexample, enols and ketones are tautomers because they are rapidlyinterconverted by treatment with either acid or base. Another example oftautomerism is the acid- and nitro-forms of phenylnitromethane, that arelikewise formed by treatment with acid or base. Tautomeric forms may berelevant to the attainment of the optimal chemical reactivity andbiological activity of a compound of interest.

As used herein a pure enantiomeric compound is substantially free fromother enantiomers or stereoisomers of the compound (i.e., inenantiomeric excess). In other words, an “S” form of the compound issubstantially free from the “R” form of the compound and is, thus, inenantiomeric excess of the “R” form. The term “enantiomerically pure” or“pure enantiomer” denotes that the compound comprises more than 95% byweight, more than 96% by weight, more than 97% by weight, more than 98%by weight, more than 98.5% by weight, more than 99% by weight, more than99.2% by weight, more than 99.5% by weight, more than 99.6% by weight,more than 99.7% by weight, more than 99.8% by weight or more than 99.9%by weight, of the enantiomer. In certain embodiments, the weights arebased upon total weight of all enantiomers or stereoisomers of thecompound.

As used herein and unless otherwise indicated, the term“enantiomerically pure R-compound” refers to at least about 95% byweight R-compound and at most about 5% by weight S-compound, at leastabout 99% by weight R-compound and at most about 1% by weightS-compound, or at least about 99.9% by weight R-compound and at mostabout 0.1% by weight S-compound. In certain embodiments, the weights arebased upon total weight of compound.

As used herein and unless otherwise indicated, the term“enantiomerically pure S-compound” or “S-compound” refers to at leastabout 95% by weight S-compound and at most about 5% by weightR-compound, at least about 99% by weight S-compound and at most about 1%by weight R-compound or at least about 99.9% by weight S-compound and atmost about 0.1% by weight R-compound. In certain embodiments, theweights are based upon total weight of compound.

In the compositions provided herein, an enantiomerically pure compoundor a pharmaceutically acceptable salt, solvate, hydrate or prodrugthereof can be present with other active or inactive ingredients. Forexample, a pharmaceutical composition comprising enantiomerically pureR-compound can comprise, for example, about 90% excipient and about 10%enantiomerically pure R-compound. In certain embodiments, theenantiomerically pure R-compound in such compositions can, for example,comprise, at least about 95% by weight R-compound and at most about 5%by weight S-compound, by total weight of the compound. For example, apharmaceutical composition comprising enantiomerically pure S-compoundcan comprise, for example, about 90% excipient and about 10%enantiomerically pure S-compound. In certain embodiments, theenantiomerically pure S-compound in such compositions can, for example,comprise, at least about 95% by weight S-compound and at most about 5%by weight R-compound, by total weight of the compound. In certainembodiments, the active ingredient can be formulated with little or noexcipient or carrier.

The compounds of this invention may possess one or more asymmetriccenters; such compounds can therefore be produced as individual (R)- or(S)-stereoisomers or as mixtures thereof.

Unless indicated otherwise, the description or naming of a particularcompound in the specification and claims is intended to include bothindividual enantiomers and mixtures, racemic or otherwise, thereof. Themethods for the determination of stereochemistry and the separation ofstereoisomers are well-known in the art.

One having ordinary skill in the art of organic synthesis will recognizethat the maximum number of heteroatoms in a stable, chemically feasibleheterocyclic ring, whether it is aromatic or non-aromatic, is determinedby the size of the ring, the degree of unsaturation and the valence ofthe heteroatoms. In general, a heterocyclic ring may have one to fourheteroatoms so long as the heteroaromatic ring is chemically feasibleand stable.

EXAMPLES

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. The examples describedin this application are offered to illustrate the compounds,compositions, materials, device, and methods provided herein and are notto be construed in any way as limiting their scope.

Materials and Methods

Lipidoid synthesis. Chemicals for lipidoid synthesis were purchased fromSigma-Aldrich and used as received. Aliphatic amine heads and eitheracrylate tails (O17O, O17S, O17Se, O18S-S, O16S-S, and N16S-S) orepoxide tails (EC18 and EC16) were mixed at 1 to 2.4 molar ratio inTeflon-lined glass screw-top vials at 70° C. for 48 h. The crudeproducts were purified using a Teledyne Isco Chromatography system.

Nanoparticle formulation. Lipidoids, cholesterol, DOPE, and DSPE-PEGwere all dissolved in ethanol solution prior to nanoparticlefabrication. For the lipidoid formulation, 16:4:1:1(Lipidoid:cholesterol:DOPE:DSPE-PEG) w/w ratio was chosen (FIG. 1A). Theethanol solution was added into triple volume of 25 mM sodium acetatebuffer (pH 5.2) drop by drop. Formulated nanoparticles were dialyzed in3.5K MWCO Slide-A-Lyzer dialysis device (Thermo Fisher) for at least 2h.

Human primary CD8+ T cells isolation. Human peripheral blood mononuclearcells (PBMCs) were purchased from Research Blood Components, LLC.Lymphocytes were isolated by density gradient centrifugation withLympholyte-H (Cedarlane) and washed with PBS. Red blood cells were lysedusing RBC Lysis Buffer (Multi-species) (eBioscience) and washed withPBS. CD8+ T cells were isolated using the CD8+ T cells isolation kit,Human (Miltenyi Biotec) according to the manufacturer's protocol.Purified CD8+ T cells were characterized by flow cytometry with CD3+antibody.

In vitro luciferase assay for lipidoids screening. Human primary CD8+ Tcells were seeded at 25,000 cells per well in 250 μL of serum-free RPMImedium containing 10 ng/mL Recombinant Human IL-2 (BD Biosciences),ImmunoCult Human CD3/CD28 T cell Activator (STEMCELL Technologies), and100 U/mL Pen-Strep (Gibco) in 48-well plates. FLuc mRNA was deliveredinto T cells immediately after seeding. For each well, 0.5 μg CleanCapFLuc mRNA (TriLink Biotechnologies) and 5 μg lipidoids were mixed in 50μL of 25 mM sodium acetate buffer (pH 5.2) and incubated at roomtemperature for 15 min for encapsulation. Then, mRNA/lipidoid complexwas added into cell culture medium at a final concentration of 1.7 μg/mLand 17 μg/mL, respectively. T cells were incubated at 37° C. for 6 h,then lysed to measure luciferase expression using Firefly LuciferaseAssay Kit 2.0 (Biotium) and SYNERGY H1 microplate reader (BioTek). Graphrepresents the average luminescence of experiments in triplicate witherror bar expressed as ±SD. The best delivery time and lipidoidconcentration was found to be ˜6 h and 30 μg/mL, respectively (FIGS.1B-1C).

In vivo FLuc mRNA delivery and bioluminescence. Lipidoid/mRNA complexeswere prepared as described above. 15 μg CleanCap FLuc mRNA (5 moU)(TriLink Biotechnologies) and 150 μg lipidoids were used per mouse in atotal volume of 150 μL PBS. Lipidoid/mRNA complexes were injected intoBALB/c mice intravenously. After 6 h of injection, 3 mg D-luciferin wasinjected intraperitoneally and bioluminescence was measured using anIVIS Spectrum CT Biophotonic Imager (PerkinElmer). After the bioimaging,mice were sacrificed and the luminescence from each organ was alsomeasured using the IVIS. Then, FLuc mRNA delivery efficacy into T cellswere quantified by luciferase assay. Single-cell suspensions from spleenwere collected by passing the splenocytes through 70-μm cell strainers,followed by red blood cell lysis using RBC Lysis Buffer (Multi-species)(eBioscience). Cells were washed once with PBS and CD8+ T cells werecollected using CD8a MicroBeads, mouse (Miltenyi Biotec) according tothe manufacturer's protocol. Splenocytes and other organs were lysedusing Firefly Luciferase Lysis Buffer (Biotium) and luminescenceintensity was measured using Firefly Luciferase Assay Kit 2.0 (Biotium)and SYNERGY H1 microplate reader (BioTek). Total protein amount wasquantified using Pierce BCA Protein Assay Kit (Thermo Scientific).Luciferase intensity was normalized to total protein amount in the cellsor tissues.

In vivo Cre mRNA delivery and confocal microscopy. Lipidoid/mRNAcomplexes were prepared as described above. 15 μg CleanCap Cre mRNA (5moU) (TriLink Biotechnologies) and 150 g lipidoids were used per mousein a total volume of 150 μL PBS. Lipidoid/mRNA complexes were injectedinto Ai14 mice intravenously every 5 days for twice. After 10 days offirst injection, mice were sacrificed and the spleen was collected. Forthe confocal microscopy imaging, spleen was frozen sectioned into 15 μmin depth. Slices were washed with PBS and fixed with acetone, followedby staining with fluorescent antibodies for CD3ε (APC), CD8a (APC), orF480 (APC). Fluorescent signal from tdTomato and antibodies was observedusing SP8 confocal microscope (Leica). For flow cytometry analysis,single-cell suspensions were collected from spleen by passing thesplenocytes through 100-μm cell strainers, followed by red blood celllysis using RBC Lysis Buffer (Multi-species) (eBioscience). Cells werewashed once with PBS and stained with fluorescent antibodies for CD4(APC) and CD8a (APC) in Flow Cytometry Staining Buffer (eBioscience).Fluorescent signal was measured using LSR-II flow cytometer (BDBiosciences).

Results and Discussion

Rough Screening of Lipidoids for mRNA Delivery into Human Primary CD8+ TCells

Cationic lipid-like materials (lipidoid) are synthesized in acombinatorial manner of hydrophilic amine head and hydrophobic carbontail through Michael addition reaction (FIGS. 3A and 3B).

Lipidoids that showed relatively good efficacy for nucleic acid andprotein delivery were selected, and a rough screening was conducted forluciferase mRNA delivery to primary T lymphocytes in vitro (FIG. 3C).Lipidoids were named using a naming convention in the form of R-OnX,R-NnX or R-ECn. In all cases, “R” indicates the amine number as shown inFIG. 3B (left column), “n” indicates the number of the carbon inhydrophobic tail before atom substitution, “X” indicates the heteroatomcontent in tail (0, S, Se, or disulfide), “EC” indicates lipidoids tailwas synthesized from epoxide. For the first-step rough screening, bothnon-formulated and formulated lipidoids with three excipients—i.e.,cholesterol, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and1,2-distearoyl-sn-glycero-3-phosphoethanolamine-[poly(ethyleneglycol)-2000] (DSPE-PEG) were tested. It was found that none of thelipidoid selected showed effective delivery to T lymphocytes when usedalone, but several lipidoids showed effective delivery when formulatedwith excipients described above (FIG. 3C). Among the various libraries,the library with chalcogen (O, S, Se)-containing tail showed the mosteffective delivery (FIG. 3C). Interestingly, lipidoids with amine head93 (1-(3-Aminopropyl)imidazole) constantly showed the effective deliveryin all tail variants (93-O17O, 93-O17S and 93-O17Se) (FIG. 3C).Treatment with mRNA alone showed no luminescence expression, indicatingthat the mRNA itself cannot enter cells without effective lipidoids.Moreover, even the most commonly used commercialized lipid reagent,Lipofectamine 2000 (LF2000), showed no transfection effect at all,revealing the great challenge in T cell transfection by non-viralparticles. According to the results from the rough screening, it wasfound that the lipidoids with an imidazole group (in amine head 93) havegreat potential in T lymphocyte transfection.

Synthesis and Detailed Screening of Lipidoids Containing Imidazole andImidazole Analogues

Based on the results of the rough screening, it was determined that theimidazole to be the key structure of the lipidoids for mRNA delivery toprimary T lymphocytes. New lipidoids containing imidazole or imidazoleanalogues, shown in FIG. 4A, were subsequently synthesized.

The imidazole or imidazole analogues-containing amine heads wereclassified into four groups: i) The carbon spacer between the imidazoleand amine group was modified with some carbon branch and differentlength (R: 9310-9315). ii) The different structures of carbon branchwere added to 2-imidazole position (R: 9321-9324). iii) The carbonspacer at 1-imidazole position were moved to 2-imidazole position andreplaced with the carbon chains with various length and branch (R:9331-9334). iv) The imidazole ring was replaced with some similarstructures (R: 9341-9352). Three aliphatic tail variations (O17O, O17Sand O16S-S) were used in constructing the new library through Michaeladdition reaction.

A detailed screening of this new library for luciferase mRNA delivery toprimary T lymphocytes in vitro was performed, as shown in FIG. 3 b , andthe correlation between amine head structure and delivery efficacy wasinvestigated. Generally, amine head 9313, 9322, 9331, as well as 93constantly showed high luminescence expression in lipidoid library.Moreover, there were some trends found in the correlation betweenchemical structures of amine heads and delivery efficacy. First, whenconsidering the linker branching structure, we found that linkers with asingle branch (e.g., 9312 and 9313) showed better efficacy than eitherthe straight-chain linker (e.g., 93, 9310 and 9315) or complex branchedlinker (e.g., 9314 and 9316). Linker length also appeared todramatically influence delivery efficacy: the 4-carbon 9315 was not aseffective as the 3-carbon 93. Similarly, the 9311 head, which isstructurally similar to 9313 but only one carbon shorter, showed nodelivery effect at all, while 9313 was extremely effective. Second, itwas found that branching at the 2-imidazole position does not affect thedelivery potency (e.g., 9322, 9323 and 9324). Third, we noted thatwithin the group with the linker at the 2-imidazole position (e.g.,9331, 9332, 9333 and 9334), all amine heads had some positive signal,indicating that linker position can be ortho-substituted and the branchon imidazole itself does not affect the delivery effect. Last, the amineheads with the imidazole ring analogues (e.g., 9341, 9351, 9352) did notdemonstrate any delivery efficacy at all, which proved the imidazolering is a key structure in T cell delivery of mRNA. In addition to theamine head structure, carbon tail structure also affected the deliveryefficacy. Among the effective amine heads (i.e., 9313, 9322, 9331 and93), lipidoids with O170 and O17S tails tail showed more than 5 fold and6 fold higher delivery efficacy than lipidoids with O16S-S tail,respectively (FIG. 4B). From these results, it was found that lipidoidssynthesized from amine head 9313, 9322, 9331 and 9332 are effective formRNA delivery to T lymphocytes, and used these amine heads for carbontail screening.

Detailed Screening of Carbon Tail Structure and Delivery Efficacy

As shown in FIG. 4B, the tails of lipidoids were the other importantfactor in the delivery efficacy. In order to further study the influenceof tail structures on T cell transfection, the detailed lipidoid librarywith 8 different carbon tails (FIG. 5A) using amine head 9313, 9322,9331 and 9332 were synthesized, which had high transfection efficacy inprevious screening.

As shown in FIG. 4B, lipidoids with O17O, O17S and O18S-S tailconsistently worked more efficiently than other tails. Interestingly,for all lipidoids using the O17C tail, in which the entire tail iscomposed of carbon without any heteroatom, delivery efficacy wassignificantly reduced. In addition, the length of the carbon chain alsoaffects delivery efficacy. Tail length of 18 carbons, with two of themsubstituted to S-S (O18S-S), worked significantly more effectively thantail length of 16 (O16S-S). Furthermore, tail with ester bond (O16S-S)worked significantly better than tail with amide bond (N16S-S). Tailsmade from epoxide (ECn series) did not show effective delivery. In orderto elucidate the positive percentage of successfully transfected Tcells, EGFP mRNA with 93-O17S and 9322-O17S was delivered into CD8+ Tcells in vitro, and quantified GFP expression with flow cytometry.Delivery efficacy into CD8+ T cells reached 7.1% with 93-O17S and 11.1%with 9322-O17S (FIG. 6 ).

The possible mechanism of the structure-related difference of mRNAdelivery to T cells might be related with the various behaviors of LNPs,such as apparent pKa values and membrane disruption abilities. In thisstudy, to further elucidate possible mechanism of the different deliveryeffect among these lipidoids, the apparent pKa value and phospholipidsbilayer membrane disruption ability were further analyzed. There was nobig difference in the pKa between effective and ineffective lipidoids,indicating the pKa might not be the main factor determining the deliveryefficacy (FIG. 7A).

However, as shown in FIG. 7B, most of the successful lipidoids showedhigher membrane disruption ability than the unsuccessful ones,especially in these lipidoids with heads in group ii).

In Vivo mRNA Delivery and Biodistribution of Nanoparticles.

Even though the in vitro screening proved that the lipidoids found fromthe structure-based screening can efficiently deliver mRNA into primaryT cells, the in vivo delivery effect would be more important for in situprogramming of T cells in human body. Moreover, owing to the complex invivo conditions, the delivery effect in the body mostly may not beconsistent with in vitro results. To investigate whether the lipidoidsidentified by in vitro screening also work in vivo, the effect oflipidoid delivery was also evaluated by intravenous injection of FLucmRNA and lipidoid complex into BALB/c mouse. 6 h after the injection,bioluminescence was detected in the live mouse, and subsequently themouse was sacrificed, and bioluminescence specifically from the heart,liver, spleen, lung and kidney was observed. All bioluminescence imagingwas performed using an In Vivo Imaging System (IVIS). Amine head 93 and9322 with O170 and O17S tails showed strong and specific luminescenceexpression in spleen (FIGS. 8A and 8B).

In the mice delivered with 9322-O17S, the luminescence intensity inspleen was 1.6 folds higher than that in liver. On the other hand, aminehead 9313, 9331, and 9332 with O17O, O17S, or O18S-S tails showed nosignal in any organs (FIGS. 8A-8C and FIGS. 9A-9B).

Mice were sacrificed and splenocytes were collected to purify CD8+ Tcells with magnetic beads. Luminescence expression from eithersplenocytes or each organ was quantified. Although there was alsoluminescence signal from liver and other types of cells in spleen, weconfirmed the effective delivery into CD8+ T cells using 93-O17S,9322-O170 and 9322-O17S (FIG. 8C). The luminescence intensity alsocorresponded to the IVIS results (FIGS. 8B and 8C).

Lipidiods Enhanced Cre Recombinase-Mediated Gene Recombination of SpleenT Cells In Vivo

In vivo delivery of gene editing molecules including mRNA, plasmid andRNP into T cells has great potential for application in T cellengineering. The potency of lipidoids for in vivo gene recombination byintravenously delivering Cre recombinase mRNA and lipidoids complex intoAi14 mice (The Jackson Laboratory) was demonstrated. The Ai14 mouse hasa genetically integrated STOP codon flanked by loxP sites located justupstream of a red fluorescent protein (tdTomato) gene 22. The STOP codoncan be removed via the activity of Cre protein, which excises the DNAfound between the loxP sites. Therefore, red fluorescence signal ismediated by the successful transfection of Cre mRNA, followed byexpression of Cre protein. Cre mRNA/lipidoid complex was injected twice,every 5 days, followed by the analysis of mouse spleen with confocalmicroscopy. Strong tdTomato expression in spleen was observed from themouse injected with either 93-O17S or 9322-O17S (FIGS. 10A and 10B),whereas no tdTomato signal was observed from the mouse injected with9313-O18S-S or PBS alone (FIG. 9C).

Spleen tissue was labeled with either CD3ε or F4/80 antibody to analyzethe localization of tdTomato signal with T lymphocytes or macrophages,respectively. In addition, CD8a antibody was used to specifically labelCD8+T lymphocytes. Yellow signal in the fluorescent image indicates thecolocalization of the red tdTomato signal and green-tagged cell-typeantibodies (indicated by the white arrows) (FIGS. 10A-10B and FIGS.8A-8C).

Single cell suspension was collected from mouse spleen and tdTomatoexpression was quantified by the flow cytometry. Both 93-O17S and9322-O17S showed significant expression of tdTomato signal compared tothe untreated control. 93-O17S reached ˜8.2% of delivery efficacy intoCD4+ T cells and ˜6.5% into CD8+ T cells in vivo (FIG. 10C and FIGS.10A-10C).

INCORPORATION BY REFERENCE

All U.S. and PCT patent publications and U.S. patents mentioned hereinare hereby incorporated by reference in their entirety as if eachindividual patent publication or patent was specifically andindividually indicated to be incorporated by reference. In case ofconflict, the present application, including any definitions herein,will control.

Other Embodiments

Those skilled in the art will recognize or be able to ascertain using nomore than routine experimentation many equivalents to the specificembodiments described herein. The scope of the present embodimentsdescribed herein is not intended to be limited to the above Description,but rather is as set forth in the appended claims. Those of ordinaryskill in the art will appreciate that various changes and modificationsto this description may be made without departing from the spirit orscope of the present invention, as defined in the following claims.

We claim:
 1. A compound of formula i:

or a pharmaceutically acceptable salt thereof, wherein R⁵ is—W-L-R^(Lipid), hydrogen, halogen, amino, hydroxyl, alkoxy, cyano,nitro, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, orheteroaryl; wherein one and only one of R⁵ is —W-L-R^(Lipid). L is adivalent linker; W is NR²⁰, O, or S; R^(Lipid) is independentlysubstituted or unsubstituted C₁₋₂₀ alkyl, substituted or unsubstitutedC₁₋₂₀ alkenyl, substituted or unsubstituted C₁₋₂₀ alkynyl, substitutedor unsubstituted C₁₋₂₀ heteroalkyl, substituted or unsubstituted C₁₋₂₀heteroalkenyl, or substituted or unsubstituted C₁₋₂₀ heteroalkynyl; andR²⁰ is R^(Lipid), H, C₁₋₆ alkyl, C₁₋₆ alkenyl, or C₁₋₆ alkynyl.
 2. Thecompound of claim 1, wherein W is NR²⁰ or S.
 3. The compound of claim 2,wherein W is S.
 4. The compound of claim 2, wherein W is NR²⁰.
 5. Thecompound of claim 4, wherein R²⁰ is R^(Lipid).
 6. The compound of anyone of claims 1-5, wherein R^(Lipid) is represented by formula II:

wherein R¹ and R² are independently H, methyl, OH, NHR³⁰, or SH; R³ andR⁴ are both H; or R³ and R⁴ are taken together to form an oxo (═O)group; Z is O, NR³⁰, or S; X and Y are independently CH₂, NR³⁰, O, S, orSe; m is an integer selected from 1-3; n is an integer selected from1-14; p is 0 or 1; q is an integer selected from 1-10; t is 0 or 1; andR³⁰ is H, C₁₋₆ alkyl, C₁₋₆ alkenyl, or C₁₋₆ alkynyl.
 7. The compound ofany one of claims 1-6, wherein R³ and R⁴ are both H.
 8. The compound ofany one of claims 1-6, wherein R³ and R⁴ are taken together to form anoxo (═O) group.
 9. The compound of any one of claims 1-8, wherein p is0.
 10. The compound of any one of claims 1-8, wherein p is
 1. 11. Thecompound of any one of claims 1-10, wherein Z is O, or NR³⁰.
 12. Thecompound of claim 11, wherein Z is O.
 13. The compound of claim 11,wherein Z is NR³⁰.
 14. The compound of claim 1, wherein the compound isa compound of formula III:


15. The compound of any one of claims 1-14, wherein R¹ and R² areindependently H, methyl, or OH.
 16. The compound of claim 15, wherein R¹and R² are both H.
 17. The compound of claim 15, wherein R¹ is H and R²is methyl.
 18. The compound of claim 15, wherein R¹ is H; and R² is OH.19. The compound of any one of claims 1-18, wherein X and Y areindependently CH₂ or O.
 20. The compound of claim 19, wherein X and Yare both CH₂.
 21. The compound of claim 19, wherein X and Y areindependently CH₂ or O and X and Y are not the same.
 22. The compound ofany one of claims 1-18, wherein X and Y are independently CH₂ or S. 23.The compound of claim 22, wherein X and Y are both S.
 24. The compoundof claim 22, wherein X and Y are independently CH₂ or S and X and Y arenot the same.
 25. The compound of any one of claims 1-24, wherein m is 1or
 2. 26. The compound of claim 25, wherein m is
 1. 27. The compound ofany one of claims 1-26, wherein n is an integer selected from 4-12. 28.The compound of claim 27, wherein n is an integer selected from 6-10.29. The compound of any one of claims 1-28, wherein q is an integerselected from 2-8.
 30. The compound of claim 29, wherein q is an integerselected from 4-8.
 31. The compound of any one of claims 1-30, wherein tis
 0. 32. The compound of any one of claims 1-30, wherein t is
 1. 33.The compound of any one of claims 1-32, wherein L is substituted orunsubstituted C₁₋₆ alkylene, substituted or unsubstituted C₁₋₆alkenylene, or substituted or unsubstituted C₁₋₆ alkynylene, substitutedor unsubstituted C₁₋₆ heteroalkylene, substituted or unsubstituted C₁₋₆heteroalkenylene, or substituted or unsubstituted C₁₋₆ heteroalkynylene.34. The compound of claim 33, wherein L is substituted or unsubstitutedC₁₋₆ alkylene.
 35. The compound of claim 34, wherein L is unsubstitutedC₁₋₆ alkylene.
 36. The compound of claim 34, wherein L is C₁₋₆ alkylenesubstituted by C₁₋₆ alkyl.
 37. The compound of claim 33, wherein L isselected from the group consisting of


38. The compound of any one of claims 1-37, wherein R⁵ is C₁₋₆ alkyl,C₂₋₆ alkenyl, C₂₋₆ alkynyl.
 39. The compound of claim 38, wherein R⁵ isC₁₋₆ alkyl.
 40. The compound of claim 1, wherein

is selected from the group consisting of:


41. The compound of claim 40, wherein

is selected from the group consisting of:


42. The compound of any one of claims 1-41, wherein each instance ofR^(Lipid) is independently selected from the group consisting of


43. The compound of claim 42, wherein each instance of R^(Lipid) isindependently selected from the group consisting of


44. The compound of claim 1, wherein

is selected from the group consisting of:

and each instance of R^(Lipid) is independently selected from the groupconsisting of


45. A lipidoid nanoparticle, comprising a compound of any one of claims1-44.
 46. The lipidoid nanoparticle of claim 45, wherein the lipidoidnanoparticle further comprises cholesterol.
 47. The lipidoidnanoparticle of claim 45 or 46, further comprising DOPE or PEG2K-DEPC.48. The lipidoid nanoparticle of any one of claims 45-47, furthercomprising a divalent nickel, wherein the compound chelates with thedivalent nickel.
 49. The lipidoid nanoparticle of any one of claims45-48, further comprising a protein or a nucleic acid.
 50. The lipidoidnanoparticle of claim 49, wherein the protein or the nucleic acid isGFP-Cre or CRISPR/Cas9.
 51. The lipidoid nanoparticle of claim 50,wherein the protein or the nucleic acid is GFP-Cre.
 52. The lipidoidnanoparticle of claim 50, wherein the protein or the nucleic acid isCRISPR/Cas9.
 53. The lipidoid nanoparticle of any one of claims 48-52,wherein the divalent nickel binds to the protein or the nucleic acid viaa non-covalent interaction.
 54. The lipidoid nanoparticle of any one ofclaims 45-53, further comprising a small molecule.
 55. The lipidoidnanoparticle of claim 54, wherein the small molecule is an antifungalagent or a chemotherapeutic agent.
 56. The lipidoid nanoparticle ofclaim 54, wherein the small molecule is selected from the groupconsisting of Bortezomib, Imatinib, Gefitinib, Erlotinib, Afatinib,Osimertinib, Dacomitinib, Daunorubicin hydrochloride, cytarabine,Fluorouracil, Irinotecan Hydrochloride, Vincristine Sulfate,Methotrexate, Paclitaxel, Vincristine Sulfate, epirubicin, docetaxel,Cyclophosphamide, Carboplatin, Lenalidomide, Ibrutinib, Abirateroneacetate, Enzalutamide, Pemetrexed, Palbociclib, Nilotinib, Everolimus,Ruxolitinib, epirubicin, pirirubicin, idarubicin, valrubicin, amrubicin,Bleomycin, phleomycin, dactinomycin, Mithramycin, streptozotecin,pentostatin, Mitosanes mitomycin C, Enediynes calicheamycin, Glycosidesrebeccamycin, Macrolide lactones epotihilones, ixabepilone, pentostatin,Salinosporamide A, Vinblastine, Vincristine, Etoposide, Teniposide,Vinorelbine, Docetaxel, Camptothecin, Hycamtin, Pederin, Theopederins,Annamides, Trabectedin, Aplidine, and Ecteinascidin 743 (ET743).
 57. Thelipidoid nanoparticle of claim 54, wherein the small molecule isAmphotericin B or Doxorubicin.
 58. The lipidoid nanoparticle of any oneof claims 45-57, wherein the lipidoid nanoparticle has a particle sizeof about 25 nm to about 1000 nm.
 59. The lipidoid nanoparticle of claim58, wherein the lipidoid nanoparticle has a particle size of about 50 nmto about 750 nm.
 60. A pharmaceutical composition, comprising a lipidoidnanoparticle of any one of claims 45-59, and a pharmaceuticallyacceptable carrier or excipient.